We describe a detailed structure determination of biosurfactant produced by Pseudozyma hubeiensis SY62, which was newly isolated from Calyptogena soyoae (deep-sea cold-seep clam, Shirouri-gai) at 1156 m in Sagami bay. P. hubeiensis SY62 was taxonomically slightly different from the P. hubeiensis type strain, which produces biosurfactants. Glycolipid production by the strain was also slightly different from those of previously reported strains. BS productivity was estimated to be around 30 g/l from the weight of the crude extract. At least five different spots of glycolipid biosurfactants (BSs) were detected by TLC. Results of nuclear magnetic resonance spectroscopies indicated the major product, namely MEL-C (4-O-[4′-O-acetyl-2′,3′-di-O-alka(e)noil-β-d-mannopyranosyl]-d-erythritol), as a promising BS. By further structural determination, the major fatty acids of MEL-C were estimated to be saturated C6, C10, and C12 acids, which were shorter than those of previously reported MEL-C. Furthermore, 1H-NMR spectra implied the presence of C2 acids as acyl groups. According to surface tension determination, the novel MEL-C showed larger critical micelle concentration (1.1 × 10− 5 M) than conventional MEL-C which bound C10 and C12 acids (9.1 × 10− 6 M). From these results, shorter fatty acids would confer hydrophilicity onto the novel MEL-C.